Vasculogenesis, the de novo formation of blood vessels, and angiogenesis, the growth of new capillaries from pre-existing ones, are critical for embryonic development and normal physiological functions in adults [Carmeliet, P., Mechanisms of angiogenesis and arteriogenesis. Nat Med, 2000 6(4) 389-95]. Failure of these processes leads to early death of the embryo as a result of impaired formation of the vascular tree and somatic growth. In adults, abnormal angiogenesis can lead to impaired wound healing, poor tissue regeneration in ischemic conditions, cyclical growth of the female reproductive system, and tumor development [Carmeliet, P. and R. K. Jain, Angiogenesis in cancer and other diseases. Nature, 2000 407(6801) 249-57].
The vascular endothelial growth factor (VEGF) family of growth factors are the most important players involved in physiological and pathological angiogenesis. Thus far, five VEGF family members have been discovered, including VEGF-A, VEGF-B, VEGF-C, VEGF-D and PlGF [Li, X. and U. Eriksson, Novel VEGF family members: VEGF-B, VEGF-C and VEGF-D. Int J Biochem Cell Biol, 2001 33(4) 421-6]. Among them, VEGF-A is the most potent angiogenic factor, but it requires fine-tuned control of its expression and regulation. Lack of a single VEGF allele results in embryonic lethality [Carmeliet P, et al., Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature, 1996 380(6573) 435-39; and Ferrara N, et al., Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature, 1996 380(6573) 439-42]. VEGF-A binds to four receptors, VEGFR-1, VEGFR-2, neuropilin-1 and neuropilin-2 [Poltorak, Z., T. Cohen, and G. Neufeld, The VEGF splice variants: properties, receptors, and usage for the treatment of ischemic diseases, Herz, 2000 25(2) 126-9]. Through these receptors, VEGF-A promotes endothelial cell proliferation, induces vascular permeability and chemo-attracts monocytes. VEGF-A expression is efficiently upregulated by hypoxia. The potent angiogenic capacity of VEGF-A gives it potential therapeutic utility in ischemic diseases where physiological angiogenesis is needed. However, clinical use of VEGF-A has been hampered because of its severe side effects [Carmeliet, P., VEGF gene therapy: stimulating angiogenesis or angioma-genesis?, Nat Med, 2000 6(10) 1102-03].
VEGF-B was the third member of the VEGF family to be discovered (after VEGF-A and VEGF-C) [Olofsson B, et al., Vascular endothelial growth factor B, a novel growth factor for endothelial cells. Proceedings of the National Academy of Sciences of the United States of America, 1996 93(6) 2576-81], [Grimmond S, et al., Cloning and characterization of a novel human gene related to vascular endothelial growth factor. Genome Research, 1996, 6(2) 124-31]. VEGF-B has similar angiogenic and other properties to those of VEGF-A, but is distributed and expressed in tissues differently from VEGF-A. In particular, VEGF-B is very strongly expressed in the heart, and only weakly in the lungs, whereas the reverse is the case for VEGF-A [Olofsson, B. et al, Proc. Natl. Acad. Sci. USA 1996 93 2576-2581]. RT-PCR assays have demonstrated the presence of VEGF-B mRNA in melanoma, normal skin, and muscle. This suggests that VEGF-A and VEGF-B, despite the fact that they are co-expressed in many tissues, have functional differences. A comparison of the PDGF/VEGF family of growth factors reveals that the 167 amino acid isoform of VEGF-B is the only family member that is completely devoid of any glycosylation. Gene targeting studies have shown that VEGF-B deficiency results in mild cardiac phenotype, and impaired coronary vasculature [Bellomo et al, Circ Res, 2000 86 E29-35].
Human VEGF-B was isolated using a yeast co-hybrid interaction trap screening technique by screening for cellular proteins which might interact with cellular retinoic acid-binding protein type I (CRABP-I). The isolation and characteristics including nucleotide and amino acid sequences for both human and murine VEGF-B are described in detail in PCT/US96/02957, in U.S. Pat. Nos. 5,840,693 and 5,607,918 by Ludwig Institute for Cancer Research and The University of Helsinki and in Olofsson et al, Proc. Natl. Acad. Sci. USA 1996 93 2576-2581. The nucleotide sequence for human VEGF-B is also found at GenBank Accession No. U48801. The entire disclosures of the International Patent Application PCT/US97/14696 (WO 98/07832), U.S. Pat. Nos. 5,840,693 and 5,607,918 are incorporated herein by reference.
The human and murine genes for VEGF-B are almost identical, and both span about 4 kb of DNA. The genes are composed of seven exons, and their exon-intron organization resembles that of the VEGF-A and PlGF genes [Grimmond et al, Genome Res, 1996 6 124-131; Olofsson et al, J. Biol. Chem. 1996 271 19310-17; Townson et al, Biochem. Biophys. Res. Commun. 1996 220 922-928].
VEGF-B binds specifically to VEGFR-1 [Olofsson B, et al., Vascular endothelial growth factor B, a novel growth factor for endothelial cells, Proc Nat'l Acad Sci USA 1996 93(6) 2576-81] and neuropilin-1 [Olofsson B, et al., Vascular Endothelial Growth Factor B (Vegf-B) Binds to Vegf Receptor-1 and Regulates Plasminogen Activator Activity In Endothelial Cells, Proc Nat'l Acad Sci USA, 1998 95(20) 11709-14], a receptor previously identified for collapsins/semaphorins [Soker, S., Neuropilin in the midst of cell migration and retraction, Int J Biochem Cell Biol, 2001 33(4) 433-37]. VEGF-B displays a unique expression pattern compared with other VEGF family members, with the highest expression level in the cardiac myocytes [Aase K, et al., Localization of VEGF-B in the mouse embryo suggests a paracrine role of the growth factor in the developing vasculature, Developmental Dynamics, 1999 215(1) 12-25], whereas VEGFR-1 is expressed in the adjacent endothelial cells [Aase K, et al., Localization of VEGF-B in the mouse embryo suggests a paracrine role of the growth factor in the developing vasculature. Developmental Dynamics, 1999 215(1) 12-25], and neuropilin-1 (NP-1) is expressed in both endothelium and cardiac myocytes during development [Makinen T, et al., Differential binding of vascular endothelial growth factor B splice and proteolytic isoforms to neuropilin-1. Journal of Biological Chemistry, 1999 274(30) 21217-22; and Kitsukawa T, et al., Overexpression of a membrane protein, neuropilin, in chimeric mice causes anomalies in the cardiovascular system, nervous system and limbs, Development, 1995 121(12) 4309-18]. The temporal-spatial expression patterns of VEGF-B and its receptors thus suggest both autocrine and paracrine roles of VEGF-B in the heart [Makinen T, et al., Differential binding of vascular endothelial growth factor B splice and proteolytic isoforms to neuropilin-1, J. Biol. Chem. 1999 274(30) 21217-22]. VEGF-B heterodimerizes with VEGF-A when co-expressed [Olofsson B, et al., Vascular endothelial growth factor B, a novel growth factor for endothelial cells, Proc. Nat'l. Acad. Sci. USA 1996 93(6) 2576-81]. Two differently spliced VEGF-B isoforms exist, VEGF-B186 and VEGF-B167, with the first isoform accounting for about 80% of the total VEGF-B transcripts [Li, X. et al, Growth Factor 2001 19 49-59]. The two polypeptides differ at their carboxy-termini and display different abilities to bind neuropilin-1 [Makinen et al., J. Biol. Chem. 1999 274(30) 21217-22]. Moreover, VEGF-B186 is freely secreted, while VEGF-B167 is secreted but largely cell-associated, implying that the functional properties of the two proteins may be distinct. Both isoforms bind to extracellular matrix tenascin-X and stimulate endothelial cell proliferation through VEGF-receptor-1 (VEGFR-1) [Ikuta, T., H. Ariga, and K. Matsumoto, Extracellular matrix tenascin-X in combination with vascular endothelial growth factor B enhances endothelial cell proliferation, Genes Cells, 2000 5(11) 913-927].
The capillary density in mice lacking VEGF-B is the same as in normal mice. However, gene targeting studies have shown that VEGF-B deficiency results in an atrial conduction abnormality characterized by a prolonged PQ interval and impaired coronary vasculature [Aase K. et al, Circulation 2001 104 358-64; WO 98/36052; and Bellomo D, et al., Mice lacking the vascular endothelial growth factor-B gene (Vegfb) have smaller hearts, dysfunctional coronary vasculature, and impaired recovery from cardiac ischemia. Circulation Research, 2000 86(2) E29-E35]. Thus, accumulating data suggest that VEGF-B has important roles in both physiological and pathological conditions in the cardiovascular system.
VEGF-B may also be involved in tumor development. VEGF-B mRNA can be detected in many tumors and most tumor cell lines [Gunningham, S. P., et al., VEGF-B expression in human primary breast cancers is associated with lymph node metastasis but not angiogenesis, J Pathol, 2001 193(3) 325-32; Andre, T., et al., Vegf, Vegf-B, Vegf-C and their receptors KDR, FLT-1 and FLT-4 during the neoplastic progression of human colonic mucosa, Int J Cancer, 2000 86(2) 174-81; Eggert, A., et al., High-level expression of angiogenic factors is associated with advanced tumor stage in human neuroblastomas, Clin Cancer Res, 2000 6(5) 1900-08; Niki, T., et al., Expression of vascular endothelial growth factors A, B, C, and D and their relationships to lymph node status in lung adenocarcinoma, Clin Cancer Res, 2000 6(6) 2431-9; and Salven P, et al., Vascular Endothelial Growth Factors Vegf-B and Vegf-C Are Expressed In Human Tumors, Am. J. Pathology, 1998 153(1) 103-108]. VEGF-B expression is especially up-regulated in tumor-associated macrophages in ovarian epithelial tumors [Sowter H., et al., Expression and Localization Of the Vascular Endothelial Growth Factor Family In Ovarian Epithelial Tumors, Laboratory Invest. 1997 77(6) 607-14] and renal cell carcinomas [Gunningham, S. P., et al., Vascular endothelial growth factor-B and vascular endothelial growth factor-C expression in renal cell carcinomas: regulation by the von Hippel-Lindau gene and hypoxia, Cancer Res, 2001 61(7) 3206-11].
Acute and chronic myocardial ischemia are the leading causes of morbidity and mortality in the industrialized society caused by coronary thrombosis [Varbella, F., et al., Subacute left ventricular free-wall rupture in early course of acute myocardial infarction. Clinical report of two cases and review of the literature, G Ital Cardiol, 1999 29(2) 163-70]. Immediately after heart infarction, oxygen starvation causes cell death of the infarcted area, followed by hypertrophy of the remaining viable cardiomyocytes to compensate the need of a normal contractile capacity [Heymans S, et al., Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure, Nat. Med., 1999 5(10) 1135-42]. Prompt post-infarction reperfusion by blood of the infarcted left ventricular wall may significantly reduce the early mortality and subsequent heart failure by preventing apoptosis of the hypertrophied viable myocytes and pathological ventricular remodelling [Dalrymple-Hay, M. J., et al., Postinfarction ventricular septal rupture: the Wessex experience, Seminar Thorac Cardiovasc Surg, 1998 10(2) 111-16].
Promotion of angiogenesis is desirable in situations where vascularization is to be established or extended, for example after tissue or organ transplantation, or to stimulate establishment of collateral circulation in tissue infarction or arterial stenosis, such as in coronary heart disease and thromboangitis obliterans. The angiogenic process is highly complex and involves the maintenance of the endothelial cells in the cell cycle, degradation of the extracellular matrix, migration and invasion of the surrounding tissue and finally, tube formation. Because of the crucial role of angiogenesis in so many physiological processes, there is a need to develop factors which will promote angiogenesis.
Administration of growth factors such as VEGF-A and FGF-2 has been considered a possible approach for the therapeutic treatment of ischemic heart and limb disorders. However, both animal studies and early clinical trials with VEGF angiogenesis have encountered severe problems [Carmeliet, Nat Med, 2000 6 1102-3; Yancopoulos et al., Nature, 2000 407 242-8; Veikkola et al., Semin Cancer Biol 1999 9 211-20; Dvorak et al., Semin Perinatol 2000 24 75-8; Lee et al., Circulation, 2000 102 898-901]. VEGF-A stimulated microvessels are disorganized, sinusoidal and dilated, much like those found in tumors [Lee et al., Circulation 2000 102 898-901; and Springer et al., Mol. Cell 1998 2 549-559]. Moreover, these vessels are usually leaky, poorly perfused, torturous and likely to rupture and regress. Thus, these vessels have limited ability to improve the ischemic conditions of myocardium. In addition, the leakage of blood vessels induced by VEGF-A (also known as Vascular Permeability Factor) could cause cardiac edema that leads to heart failure. Unregulated VEGF-A expression in the myocardium also could lead to the development of hemangioma or the growth of micrometastases in distal organs instead of functional vessels.
Thus, despite some advances in clinical treatment and prevention which have been achieved in the prior art, insufficient or abnormal post-infarction revascularization remains a major cause of the death of the otherwise viable myocardium and leads to progressive infarct extension and fibrous replacement, and ultimately heart failure. Therefore, therapeutic agents promoting normal post-infarction revascularization with minimal toxicity are still needed and there is an ongoing requirement for new angiogenic factors and new methods of angiogenic therapy.